The present invention relates generally to sensors for nitric oxide in biological systems.
Nitric oxide (NO) has been reported to have important roles in diverse fields, ranging from neuroscience to urology and cardiovascular medicine, and was named "molecule of the year" in 1992 (Koshland (1992) 258:1861-1865). Nitric oxide is known to be produced by the endothelial lining of blood vessels and to be responsible for blood vessel dilation. In cardiovascular medicine, nitric oxide has been implicated in the diseases of hypercholesterolemia, atherosclerosis, and hypertension (Gibaldi (1993) J. Clin. Pharmacol. 33:488-496; Star (1993) Am. J. Med. Sci. 306:348-358; Tanner et al. (1993) Sem. Thrombosis Hemostasis 19:167-175). It has also been shown that large quantities of nitric oxide are produced during massive infection, and may be responsible for septic shock, a common complication of post-surgical patients.
Biological measurements of nitric oxide have been reported in cultured cells or isolated perfused organs. Nitric oxide has only been measured in human subjects indirectly, through stimulation or inhibition of substrates and enzymes involved in nitric oxide production.
Fiber optic sensors have been used to measure oxygen, carbon dioxide, and the pH of blood inside an artery. A fiber optic sensor has been used for measuring nitrogen oxide in the atmosphere; however, this sensor is not sufficiently sensitive to detect nitric oxide concentrations in biological systems.
Two types of electrochemical sensors have been described for nitric oxide. Shibuki (1990) Neurosci. Res. 9:69-76, developed an electrode based on a modified oxygen electrode for measurement of NO concentrations less than 100 nM in brain tissue. The linear range of the Shibuki electrode is between 1-3 .mu.M. Because of the frequent calibration required by oxygen electrode-type sensors, a NO electrode based on this technology is not suitable for in vivo continuous monitoring in a biological system.
Malinski and Taha (1992) Nature 358:676-678, developed a sensor which measures current or voltage resulting from the electrochemical oxidation of NO to the nitrite ion NO.sup.-2 catalyzed by a polymeric metalloporphyrin. The Malinski sensor is very small, composed of a fragile carbon fiber which allows measurement of NO in a single cell. Malinski and Taha report a detection limit of 10 nM with a linear response up to 300 .mu.M and a response time of 10 msec.
Optical methods have been previously developed for measuring NO concentration in cell cultures and perfusate from isolated organs. Chemiluminescence has been used to detect NO by its reaction with ozone (Feelisch and Noack (1987a) Eur. J. Pharmacol. 142:465-469) or luminol-H.sub.2 O.sub.2 (Kikuchi et al. (1993) Anal. Chem. 65:1794-1799) to produce light emission. Although a sensitive technique, methods of chemiluminescence detection cannot be readily adapted for use in a fiber optic sensor because they require the addition of an analyte in order to generate light.
The diazotization assay is a spectrophotometric assay which measures nitric oxide through its conversion to the nitrite ion. This assay is not suitable for measurement of NO in a biological system because it is not specific for NO since nitrites are also produced from other sources (Tracey et al. (1990) J. Pharmacol. Exp. Ther. 252:922-928). Further, the detection limit is about 0.2 to2.0 .mu.M (Tracey (1992) Neuroprotocols 1:125-131).
Suggested methods for immobilizing biomolecules include covalent binding, physical adsorption, or cross-linking to a suitable carrier matrix, and physical entrapment and microencapsulation in a polymeric or SiO.sub.2 matrix (sol-gel) (Dave et al. (1994) Anal. Chem. 66:1120A-1127A). The use of hemes and porphyrins immobilized by the sol-gel method have been investigated in the development of NO sensors. For example, Eguchi et al. (1990) Sensors and Actuators B1:154-157 investigated various metal ion-doped porphyrins immobilized onto the end of a fiber for the measurement of nitric oxide from combustion products. They found that Co-doped porphyrin was most sensitive to nitric oxide in the range of 10-1000 .mu.M at 200.degree. C. Eguchi et al. monitored the change in optical absorption at 420 nm, attributing this change to the oxidation of Co(I) and the formation of Co(II)-NO complex. Dave et al. (1994) supra report the use of sol-gel immobilized manganese myoglobin for measurement of NO.